Confocal heterodyne interference microscopy

ABSTRACT

An improved confocal microscope system is provided which images sections of tissue utilizing heterodyne detection. The system has a synthesized light source for producing a single beam of light of multiple, different wavelengths using multiple laser sources. The beam from the synthesized light source is split into an imaging beam and a reference beam. The phase of the reference beam is then modulated, while confocal optics scan and focus the imaging beam below the surface of the tissue and collect from the tissue returned light of the imaging beam. The returned light of the imaging beam and the modulated reference beam are combined into a return beam, such that they spatially overlap and interact to produce heterodyne components. The return beam is detected by a photodetector which converts the amplitude of the return beam into electrical signals in accordance with the heterodyne components. The signals are demodulated and processed to produce an image of the tissue section on a display. The system enables the numerical aperture of the confocal optics to be reduced without degrading the performance of the system.

This application is a continuation of application Ser. No. 09/086,117filed May 28, 1998 now U.S. Pat. No. 6,151,127.

FIELD OF THE INVENTION

The present invention relates to confocal microscopy for examination ofobjects, such as biological tissue, and particularly to a confocalmicroscope system for scanning below the surface of tissue, whichutilizes heterodyne detection to produce confocal images of tissuesections. This invention is especially suitable for providing aninstrument for dermal or surgical pathology applications.

BACKGROUND OF THE INVENTION

Confocal microscopy involves scanning a tissue to produce microscopicimages of a slice or section of tissue. Such microscopic imaged sectionsmay be made in-vivo and can image at cellular resolutions. Examples ofconfocal scanning microscopes are found in Milind Rajadhyaksha et al.,“In vivo Confocal Scanning Laser Microscopy of Human Skin: Melaninprovides strong contrast,” The Journal of Investigative Dermatology,Volume 104, No. 6, June 1995, pages 1-7, and more recently, in MilindRajadhyaksha et al., “Confocal laser microscope images tissue in vivo,”Laser Focus World, February 1997, pages 119-127. These systems haveconfocal optics which direct light to tissue and image the returnedreflected light. Such confocal microscope systems can focus and resolvea narrow width of tissue as an imaged section, such that tissuestructures can be viewed at particular depths within the tissue, therebyavoiding evasive biopsy procedures for pathological examination of thetissue, or allow pathological examination of unprepared excised tissue.

Two parameters which effect the performance of confocal microscopesystems in imaging tissue sections are the numerical aperture (NA) ofthe optics and the wavelength of the beam scanned through the tissue.The axial resolution, i.e., the thickness of the imaged section, andlateral resolution of confocal microscope systems are directlyproportional to the wavelength of the light source and inverselyproportional to NA² (axial) and NA (lateral). In other words, the higherthe NA, the thinner the imaged section, while the lower the NA, thethicker the imaged section. Both the axial resolution and the lateralresolution are optimized in a confocal microscope system suitable forpathological examination to the dimensions of the tissue structures,such as cells, which are of interest. As discussed in the MilindRajadhyaksha et al. article appearing in Laser Focus World, February1997, the use of a near-infrared light source between about 700 nm and1200 nm and optics with a NA of about 0.7-0.9 have provided optimalresults for imaging tissue sections with sufficient discrimination ofcellular level structures. One problem with using optics providing NAvalues about this range is that they are large and expensive,particularly for the objective lens which focuses light into andcollects light from the tissue, and are very sensitive to aberrations,such as introduced by the object being imaged. Accordingly, it isdesirable to provide imaging of tissue sections in a confocal microscopeusing lower cost and smaller optics having a NA below 0.7 withoutsacrificing imaging performance, in particular depth discrimination andscattered light rejection.

Accordingly, it is a feature of the present invention to improveconfocal microscopy by combining the depth response of confocal imagingwith the coherence function of heterodyne detection using a synthesizedbeam of multiple wavelengths of light, such that lower NA confocaloptics and inexpensive laser diode sources may be used. Heterodynedetection has been proposed for imaging in U.S. Pat. No. 5,459,570,which describes an apparatus using an optical coherence domainreflectometer for providing images of a tissue sample to perform opticalmeasurements. However, this apparatus is limited in depth resolution anddoes not utilize confocal optics for microscopic imaging. Other opticalsystems have used multiple wavelengths of light, but are limited togenerating interference patterns for visualizing fringes characterizingthe surface of objects, such as shown in U.S. Pat. No. 5,452,088, whichdescribes a multi-mode laser apparatus for eliminating backgroundinterference, and U.S. Pat. No. 4,632,554, which describes a multiplefrequency laser interference microscope for viewing refractive indexvariations. Such interferometric-based optical systems have no confocaloptics or heterodyne detection, nor do they provide imaging within atissue sample. A confocal microscope using multiple wavelengths of lighthas been proposed in U.S. Pat. No. 4,965,441, but this microscope islimited to focusing at different altitudes for surface examination of anobject and does not have heterodyne detection.

SUMMARY OF THE INVENTION

It is the principal object of the present invention to provide animproved confocal microscopy method and confocal microscope system forimaging sections of tissue using heterodyne detection.

It is another object of the present invention to provide an improvedconfocal microscope system for imaging tissue using a synthesized lightsource to produce a beam having different wavelengths, in which thesynthesized light source combines beams from multiple light sourcesproducing light at each of the different wavelengths.

It is another object of the present invention to provide an improvedconfocal microscope system for imaging which can use low NA confocaloptics, such as below 0.7, while achieving imaging performance in termsof axial resolution equivalent to prior art confocal microscope systemsusing higher NA confocal optics, such as between 0.7 and 0.9.

Briefly described, the system embodying the present invention includes asynthesized light source for producing a single beam of light ofmultiple, different wavelengths from multiple laser sources, and a firstbeam splitter for separating the single beam into an imaging beam and areference beam. The phase of the reference beam is modulated by anoptical modulator, while confocal optics scan and focus the imaging beambelow the surface of the tissue and collect returned light of theimaging beam from the tissue. A second beam splitter is provided forinteracting the returned light of the imaging beam with the modulatedreference beam to provide a combined return beam having heterodynecomponents. The heterodyne components in the return beam represent thespatial overlapping of the imaging and reference beams over thebandwidth of the different wavelengths produced by the synthesized lightsource. The return beam is received by a photodetector which convertsthe amplitude of the light of the return beam into electrical signals inaccordance with such heterodyne components representative of the tissuesection. The electrical signals are then processed by a controller, suchas a computer, to produce an image of the tissue section on a displaycoupled to the controller.

To promote the interaction of the imaging and reference beams in thereturn beam, the path lengths of the imaging and reference beams arematched such that the difference between their path lengths areapproximately equal to integer multiples of the separation of the peaksin the coherence function produced by the synthesized light source.

The performance of the system, in terms of the axial resolution of theimaged tissue section, depends on the numerical aperture (NA) of theconfocal optics and the multiple, different wavelengths of the beamproduced by the synthesized light source, such that lower NA optics canbe used to provide an axial resolution previously afforded by confocalmicroscope systems using higher NA optics between 0.7 and 0.9.

The system improves confocal microscopy by combining the axialresolution of confocal detection and the axial ranging of heterodynedetection of light with a coherence function, that is preferablyperiodic, to provide an axial (depth) resolution that is an improvementover that provided by confocal or heterodyne detection alone. It isbelieved that the heterodyne components are produced by overlapping oneof the peaks of the coherence function with the broader depth responseof the confocal optics, while all the other peaks do not contribute tothe image of the tissue due to suppression by the confocal depthresponse. The signal is spatially limited to a region of said tissue inthe focal plane along which the confocal optics scan and focus theimaging beam in the tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing objects, features and advantages of the invention willbecome more apparent from a reading of the following description inconnection with the accompanying drawings in which:

FIG. 1 is a block diagram of the system in accordance with the presentinvention;

FIG. 2 is a block diagram of the synthesized light source of FIG. 1; and

FIG. 3 is a graph showing an example of the axial resolution of thesystem of FIG. 1 in terms of depth response.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, a confocal microscope system 10 is shown forproducing images of sections of an object 12, such as a tissue sample orin-vivo tissue of patient, below the surface of the object. System 10includes a synthesized light source 14 providing a single beam 15 havingseveral, different wavelengths of light. Synthesized light source 14includes a number (N) of multiple light sources each providing lightbeams at a different wavelength, which are combined into a single beam15. Beam 15 thus represents light that has a coherence function withnarrow peaks depending on the wavelengths (or frequencies) of each ofthe multiple light sources of synthesized light source 14. Thewavelengths of the light sources of the synthesized light source areselected to be transparent to object 12 to a particular depth from theobject's surface. For tissue, such transparency occurs in the infraredspectrum of light.

An example of synthesized light source 14 with four light sources (N=4)is shown in FIG. 2 in which the light sources are represented by lasermodules 16. The four laser modules 16 each provide a beam at a differentwavelength (or band) λ₁, λ₂, λ₃, and λ₄, respectively. Preferably, thewavelengths satisfy the equation λ₄<λ₃<λ₂<λ₁ and the wavelengths areequally separate from each other, such as at 80 nm intervals in therange of 700 nm to 1200 nm. Each beam is successively combined bydichroic beam splitters 18 to provide a single beam 15 of discretewavelengths (or bands) λ₁, λ₂, λ₃, and λ₄. Specifically, the beam (λ₁)from laser modules 16 a and the beam (λ₂) from laser module 16 b arecombined by beam splitter 18 a. The beam (λ₁,λ₂) from beam splitter 18 ais then combined by a beam splitter 18 b with the beam (λ₃) from lasermodule 16 c. The beam (λ₁,λ₂, λ₃) from beam splitter 18 b is nextcombined with the beam (λ₄) from laser module 16 d to provide beam 15(λ₁,λ₂, λ₃, λ₄). Any number of beams may be combined in this manner.Each beam splitter 18 maximally transmits each wavelength, for example,beam splitter 18 c may have transmission greater than 95% for eachwavelength band λ₁,λ₂, λ₃. Similarly, each beam splitter reflectsmaximally the wavelength to be combined. For example, beam splitter 18 creflects λ₄ with reflectivity greater than 95%. The laser modules 16 mayuse diode lasers to produce a beam of high brightness which are eitherfixed, or tunable, to a desired wavelength, and have sufficiently largecoherence lengths, such as greater than 1 mm, and may be for example thefollowing commercially available laser diodes Toshiba TOLD9215, SanyoSDL3034, Sanyo SDL4032, and EG&G C86136, C86125E.

Returning to FIG. 1, beam 15 from synthesized light source 14 is splitby a beam splitter 20 into an imaging beam 22, which travels along animage arm 23 of system 10, and a reference beam 24, which travels alonga reference arm 25 of the system. Beam splitter 20 may be anon-polarizing beam splitter having similar optical transmissionproperties for each of the different wavelengths of synthesized lightsource 14, so that the merging of each wavelength is combined additivelyand linearly. For example, if synthesized light source 14 has two lightsources, beam splitter 20 would be approximately 50/50 (equallytransmissive) for the wavelength band of each source.

The imaging beam 22, passing through a beam splitter 32, is incident ona scanning device 26 for deflecting the beam 22 into a scanning beam 28that is focused by an objective lens 30 into object 12. The multiplewavelength imaging beam is thus scanned and focused to a polychromaticspot at a depth into the object (i.e., below the surface of the object).The scanning device 26 may be a typical scanning mechanism fortwo-dimensional imaging, such as a rotating polygon mirror for scanningin a first direction, and a galvomirror which deflects the beam in asecond direction. Other scanning mechanisms may also be used, such asone or two positionable galvomirrors. A relay mirror 29 may be providedto deflect the scanning beam to objective lens 30.

The imaging beam is returned from the surface or internal section of theobject 12 to be imaged. Objective lens 30 collects the reflected lightof the imaging beam 22 from the object 12, and such collected light isdeflected via relay mirror 29 and scanning device 26 to beam splitter32.

The reference beam 24 is transmitted, via relay mirror 27, to a phasemodulator 34, such as an acousto-optic modulator, which is operated by asignal from a CW oscillator 35, to modulate beam 24, and hence eachwavelength of the beam, by a certain frequency produced by oscillator35, such as 100 MHz. The frequency of the phase modulation does not varyduring scanning by scanning device 26, and should be greater than thescan frequency, i.e., the velocity of the scanning beam at the objectdivided by the lateral resolution. Alternatively, the acousto-opticmodulator may be replaced by a piezo-actuated moving mirror whichproduces a frequency (Doppler) shift in the reference beam, or by anelectro-optical modulator. The modulated reference beam 24 a isdeflected, via relay mirror 31, to a beam splitter 32. At beam splitter32, the modulated reference beam 24 a and the imaging beam lightreturning from the object 12 are combined into a return beam 36, so thatthe modulated reference beam and the imaging beam returned lightspatially overlap and interact to produce heterodyne components in thereturn beam 36. Beam splitter 32 may be a non-polarizing beam splitterhaving similar optical transmission properties for the differentwavelength bands of light from synthesized light source 14.

The return beam 36 is incident on a photodetector 38. Photodetector 38may consist of a single photo-diode detector 37 a and a confocalaperture 37 b, such as a pinhole, in the path of return beam 36.Aperture 37 b reduces the amount of stray light that falls on detector37 a. It is believed that spatial limiting (filtering) detection to aparticular region in the focal plane of the scanned imaging beam may beprovided by the use of heterodyne detection in which the reference beamacts as an aperture for the returned light of the imaging beam from theobject. Alternatively, or in combination, aperture 37 b provides forsuch spatial filtering. Photodetector 38 may also be accomplished by alens (not shown) which has a focal power which overfills photo-diode 37a, instead of confocal aperture 37 b. The synthesized light source 14,beam splitters 20 and 32, scanning device 26, objective lens 30, andphotodetector 38, represent the confocal optics of system 10, and may besimilar to those used by a typical confocal imaging system, except thata synthesized light source is used rather than conventionalmonochromatic light source illumination.

Photodetector 37 a of the photodetector assembly 38 produces anelectrical signal 39 in response to the amplitude of the light incidentupon the photodetector in the range of the different wavelengths of thesynthesized light source. Signal 39 is inputted to a demodulator 40,which operates at the same modulating frequency as phase modulator 34 todemodulate the signal, such that the electrical output signal 42 fromdemodulator 40 is directly proportional to the amplitude of themodulation in signal 39. Demodulator 40 may operate via an input signalfrom oscillator 35, if needed, however the demodulator is not sensitiveto the phase of signal 39.

For example, demodulator 40 may operate by typical amplitudedemodulation (similar to demodulation of an AM radio), or demodulationas described in U.S. Pat. No. 5,459,570.

A controller 44 is provided in system 10 which receives the outputsignal 42 from the demodulator and generates an image on a display (orCRT) 46 responsive to signal 42 as successive frames in real-time, inaccordance with the scanning pattern of scanning device 26. For example,controller 44 may sample signal 42 to acquire data represent successiveraster lines of an image correlated to the scanning mechanism as itscans the imaging beam successively across the object. Controller 44 mayalso enable and disable the operation of system 10 by controlling thelight source 14, scanning device 26, phase modulator 34, oscillator 35,and demodulator 40, via control lines not shown. Optionally, theposition of the scanning device 26 during the scan may be monitored orcontrolled by the controller 44. The controller may be a computer, suchas a PC, which uses typical display driving software for producingimages on display 46 coupled to the computer.

In system 10, the imaging beam 22 and reference beam 24 desirably areboth beams of collimated light. This may be provided by assuring thatbeam 15 from synthesized laser source 14 is collimated by the opticsahead of the beam splitter 20 before being split into the imaging andreference beams. If needed, a collimation telescope or lens may be usedwith the beam from each of the light sources of the synthesized lightsource 14 to achieve such collimation of beam 15. By using collimatedlight in system 10, the imaging and reference beam waves will have thesame phase curvature at photodetector 37 a, such that they spatiallyoverlap and interact (by interference) with each other in the returnbeam 36 at the photodetector 37 a to produce heterodyne components.

The imaging beam travels along the image arm path which represents thepath of the imaging beam 22 from beam splitter 20 to object 12 and thereturned light from the object to beam splitter 32, while the referencebeam travels along the reference arm path which represents the path frombeam splitter 20 through modulator 34 to beam splitter 32. The lengthsof the reference arm path and the image arm path are matched in system10 so that the imaging beam and reference beam interact to produceheterodyne components. Matching of the reference arm and image arm pathsoccurs when the difference in length of the two paths are approximatelyequal to integer multiples of the separation of the peaks of thecoherence function of the synthesized light source 15. This separationis dependent on each of the different wavelengths of light produced bythe synthesized light source. In the case of two light sources withinsynthesized light source 14, correction of any mismatch between thereference and image arm paths lengths may be achieved by turning off onelaser source first (which gives the confocal response) and then, withboth laser sources on, adjusting the reference arm length using theconfocal image on display 46 as a template. With more that twowavelengths the above procedure can be repeated for each wavelengthused.

The performance of system 10 in terms of axial resolution of the imagedtissue section depends on the number of light sources in the synthesizedlight source 14, the wavelengths of such light sources, and the NA ofthe confocal optics of the system. Table I below shows an example of theperformance of system 10 using the combination of any two (N=2) of fivedifferent light sources at wavelengths 670 nm, 780 nm, 830 nm, 905 nm,and 1050 mn, respectively. For each pair of wavelengths in a column androw of Table I, the optimum (or actual) NA of the confocal optics foruse with such wavelengths is first indicated, and then the benefit inaxial resolution provided is shown by the arrow to the NA which would berequired of the confocal optics to obtain such axial resolution at thelower of the two wavelengths.

TABLE I λ (nm) 670 780 830 905 1050 670 x 0.53 → 0.69 0.62 → 0.79 0.71 →0.87 0.83 → 0.95 780 x 0.35 → 0.48 0.52 → 0.69 0.71 → 0.87 830 x 0.40 →0.55 0.64 → 0.81 905 x 0.52 → 0.69 1050 x

For example, using two light sources in synthesized light source 14which operate at wavelengths 670 nm and 830 nm, respectively, andconfocal optics having an NA of 0.62, the axial resolution provided bythe system is the same as if such confocal optics had an NA of 0.79 andthe synthesized light source where substituted for a single light sourceat 670 nm. Optimal results in Table I, where the actual NA is the lowestand provides performance near the NA range of 0.7 to 0.9, occurs atcombinations of wavelengths 670 nm and 780 nm, 780 nm and 905 nm, and905 nm and 1050 nm. Accordingly, lower NA confocal optics can be used toproduce the same axial resolution afforded by higher NA confocal opticsin prior art single light source confocal microscope systems.Furthermore, if all five light sources of Table I were used in system10, the optimum NA of the confocal optics is 0.47 and the axialresolution provided by the depth response is equivalent using confocaloptics with an NA of 0.8 with only a single light source at 670 nm.

Preferably, only two laser sources are used to reduce the complexity ofthe system, however, more than two laser sources may be provided suchthat combinations of all or some of their wavelengths may be used toprovide the desired response of system 10. Thus, the illuminationprovided by the synthesized light source provides freedom in the choiceof the bandwidth over the different wavelengths of such illumination.

In imaging tissue sections by system 10, longitudinal chromaticaberrations may be corrected, if needed, by individually adjusting theposition of the focus for each wavelength produced by the synthesizedlight source. Spherical aberrations in the system, introduced primarilyby the sample, may be reduced by the use of index matching materialbetween the objective lens and the object, or by the use of an indexmatched immersion objective lens. The use of low NA confocal optics mayfurther reduce spherical aberration. Objective lens 30 may be either adry or immersion objective lens 30, although penetration depth of thescanning beam may be improved by the use of an immersion objective lens.

The following theoretical explanation is given in order to demonstratethe improvement obtained by means of heterodyne detection in accordancewith the invention. The presentation of the explanation does not implylimitation of the invention to any theory of operation. The explanationuses the following terms, equations and parameters presented below.

N is the number of light sources in the synthesized light source inwhich λ_(N) is the wavelength of the Nth source. For example, λ₁ is thewavelength of the first source, and λ₂ is the wavelength of the secondlight source. This explanation considers two light sources in order tosimplify the mathematics, in which the spectral intensity of the beamsprovided by each source is equal to I. λ₀ is the center wavelengthbetween the two wavelengths λ₁, and λ₂, and ω₀ is the center frequencybetween the frequency of the two sources. Δz is the axial distance ofthe object plane from the focal plane, and k is the wave vector of theillumination at the center frequency ω₀, where k=ω₀/c=2π/λ₀. a is thehalf-angle of the objective lens aperture. The numerical aperture NA isNA=nsinα. The depth response, i.e., the square of demodulated signalfrom a planar reflector as a function of Δz, is as follows:$\begin{matrix}{{S^{2}\left( {\Delta \quad z} \right)} = {I^{2}{{\frac{\sin \left\lbrack {2k\quad \Delta \quad z\quad {\sin^{2}\left( {\alpha/2} \right)}} \right\rbrack}{2k\quad \Delta \quad z\quad {\sin^{2}\left( {\alpha/2} \right)}}{\cos \left\lbrack {{\left( {\lambda_{2} - \lambda_{1}} \right)/\lambda_{0}}k\quad \Delta \quad z} \right\rbrack}}}^{2}}} & (1)\end{matrix}$

The first term of this equation represents the confocal response, whichis modified by the second term, representing the field correlationfunction provided by the synthesized light source. The maximumimprovement in the depth response occurs if the sine function of theconfocal response term and the cosine function in the field correlationterm are oscillating at the same frequency, i.e., if

(λ₂−λ₁)/λ₀=2sin²(α/2)  (2)

then, the depth response becomes $\begin{matrix}{{S^{2}\left( {\Delta \quad z} \right)} = {I^{2}{\frac{\sin \left\lbrack {4k\quad \Delta \quad z\quad {\sin^{2}\left( {\alpha/2} \right)}} \right\rbrack}{4k\quad \Delta \quad z\quad {\sin^{2}\left( {\alpha/2} \right)}}}^{2}}} & (3)\end{matrix}$

which is twofold narrower than the confocal response at λ₀, The confocalresponse at λ₀ being Equation (1) absent the second term,cos[(λ₂−λ₁)/λ₀kΔz].

Since the field correlation function modifying the confocal response inEquation (1), cos(Δλ/λ₀kΔz), is periodic, any mismatch between theoptical length of reference and image arm paths may be corrected withinthe confocal depth response, for example, within less than 20 μm. Theworst mismatch possible occurs if the length of the reference arm isΔz=3.2/(4ksin²(α/2)) away from the next optimum matching point where theinteraction of the imaging and reference beams generate heterodynecomponents.

Referring to FIG. 3, the depth response using two light sources (N=2)with equidistant wavelength separation Δλ and five light sources (N=5)in synthesized light source 14 is shown as a function of the unit u,where u=4kΔz sin²(α/2). For an odd number N of light sources withequidistant wavelength separation in synthesized light source 14, thedepth response of Equation (3) is${S^{2}(u)} = {\frac{\sin \left\lbrack {{Nu}/2} \right\rbrack}{u/2}}^{2}$

The depth response narrows as N increases, i.e., as the number ofdifferent wavelengths which are used increases. The depth response of asingle light source (N=1) is also illustrated in FIG. 3 for purposes ofshowing the narrowing depth response provided by using multiple,different wavelengths of light.

As discussed in connection with Table I, the improvement in depthresponse by using more than one light source is comparable to the effectof the increase of axial resolution by the use of higher NA confocaloptics in system 10. For example, if a single light source were used,instead of synthesized light source 14, at wavelength 820 nm withconfocal optics providing a NA of 0.2, the lateral resolution of system10 would be 3.32/(ksinα)≈2.2 μm and the axial resolution would be5.56/(4ksin²(α/2))≈18.0 μm. However, if the synthesized light source isused with two light sources at wavelengths 812 nm and 828 nm, the axialresolution of the system can be reduced in half to 9 μm using the sameconfocal optics. In this example, the reference and image arm pathlength mismatch may be corrected within a range of about ±10 μm with asensitivity of 1 μm.

The optimum NA for the confocal optics in system 10 using multiplewavelengths is determined by Equation (2) if the wavelength separationis of equal amount Δλ=λ₂−λ₁. In other words, the wavelengths are equallyseparated from each other by Δλ. For synthesized light source 14, thechoice of NA should be such that the confocal response of the opticalsystem suppresses all except one of the peaks of the coherence functionof the synthesized light source. The adjustment of the system may beeasily facilitated by the wavelengths of the synthesized light source 14being spaced equidistant from each other, thereby producing a periodiccoherence function. In this case, any peak of the coherence function canbe chosen to coincide with the peak of the confocal depth response byadjusting the arm length mismatch between image arm and reference arm.

By utilizing synthesized light source 14 for illumination of object 12and detection at photodetector 37 a of the heterodyne interaction of theimaging and reference beams, the performance of the system 10 in termsof axial resolution (and contrast) is improved beyond that limited bythe NA of the confocal optics of the system (which primarily is due tothe NA of objective lens 30). It is believed that the depthdiscrimination imposed by the temporal field correlation of thesynthesized light source 14 in combination with the axial resolution ofthe confocal optics improves the ability or resolution of the confocaloptics, enabling a user of the microscope system 10 to betterdistinguish cellular level tissue structures in the imaged section ofobject 12 on display 46.

From the foregoing description, it will be apparent that an improvedconfocal microscope system and method for confocal microscopy utilizingheterodyne detection has been provided. Variations and modifications ofthe herein described system and method and other applications for theinvention will undoubtedly suggest themselves to those skilled in theart. Accordingly, the foregoing description should be taken asillustrative and not in a limiting sense.

What is claimed is:
 1. A confocal microscope system for imaging sectionsof tissue below the surface of said tissue comprising: a light sourcefor providing a single beam of long coherence length light having aplurality of different non-overlapping wavelength bands; first opticsfor separating said single beam into an imaging beam and a referencebeam; an optical modulator for modulating the phase of said referencebeam; confocal optics for scanning and focusing said imaging beam belowthe surface of said tissue and collecting returned light of said imagingbeam from said tissue; second optics for interacting said returned lightwith said modulated reference beam to provide a combined return beamwhich has heterodyne components; a detector which receives said returnbeam and produces electrical signals corresponding to said components;and means for processing said electrical signals to produce an image ofsaid tissue section.
 2. The system according to claim 1 wherein saidsingle beam from said light source defines a coherence function havingpeaks in accordance with said different wavelength bands, and saidimaging beam transverses along a first path from said first optics tosaid tissue and from said tissue to said second optics, and saidreference beam transverses along a second path from said first opticsthrough said optical modulator to said second optics, in which thedifference in length of said first path from said second path isapproximately equal to integer multiples of the separation of the peaksin said coherence function.
 3. The system according to claim 1 whereinsaid single beam from said light source defines a coherence functionhaving peaks in accordance with said different wavelength bands, andsaid collected light of said imaging beam by said confocal opticsdefines a confocal response having a peak, in which said second opticsoverlap one of said peaks of said coherence function present in saidreference beam with said peak of said confocal response.
 4. The systemaccording to claim 1 wherein said confocal optics scan and focus saidimaging beam along a focal plane in said tissue, and said reference beamat said second optics provides for spatially limiting the light of saidreturn beam to a region of said tissue in said focal plane.
 5. Thesystem according to claim 1 wherein said confocal optics scan and focussaid imaging beam along a focal plane in said tissue, and said detectorprovides for spatially limiting the light of the return beam to a regionof said tissue in said focal plane.
 6. The system according to claim 1wherein said processing means further comprises means for demodulatingsaid electrical signals to obtain data representative of said image ofsaid tissue section.
 7. The system according to claim 1 wherein saidlight source comprises a plurality of light sources producing aplurality of different beams at each of said different wavelength bands,and third optical elements for combining said plurality of beams intosaid single beam.
 8. The system according to claim 7 wherein said imageof said tissue section has an axial resolution dependent on at least thenumber of said plurality of light sources.
 9. The system according toclaim 7 wherein said light sources are laser sources.
 10. The systemaccording to claim 9 wherein said laser sources are diode lasers. 11.The system according to claim 1 wherein said single beam of longcoherence length light has a plurality of different non-overlappingwavelength bands of coherence length greater than one millimeter. 12.The system according to claim 1 wherein said confocal optics provide anumerical aperture below 0.7.
 13. The system according to claim 1wherein said scanning and collecting means comprises a deflector forscanning said imaging beam in at least two dimensions.
 14. The systemaccording to claim 1 wherein said first and second optics each representa beam splitter.
 15. The system according to claim 1 wherein saiddifferent wavelength bands are in the infrared spectrum of light. 16.The system according to claim 1 wherein said imaging beam is scanned andfocused by said confocal optics to an illumination spot.
 17. The systemaccording to claim 1 wherein said confocal optics have a numericalaperture and the tissue section imaged has an axial resolution which isthe same as that provided by other confocal optics having a higher oneof said numerical aperture when used with a single wavelength beam. 18.The system according to claim 1 wherein said detector further comprisesa photodetector and means for spatial limiting the return beam onto saidphotodetector.
 19. The system according to claim 18 wherein said spatiallimiting means limits the returned light incident the photodetector to aparticular region of the tissue.
 20. The system according to claim 1wherein said confocal optics define a numerical aperture, and saidtissue section imaged has an axial resolution in accordance with atleast the numerical aperture of the confocal optics and the number ofdifferent beams providing said single beam.
 21. The system according toclaim 1 wherein said wavelength bands are equidistant from each other.22. The system according to claim 1 wherein said long coherence lengthlight represents light with coherence length greater than onemillimeter.
 23. A method for confocal microscopy which images sectionsof tissue comprising the steps of: producing a single beam of longcoherence length light having a plurality of different non-overlappingwavelength bands; separating said single beam into an imaging beam and areference beam; modulating the phase of said reference beam; scanningand focusing said imaging to said tissue and collecting returned lightfrom said tissue; interacting said returned light with said modulatedreference beam to provide a combined return beam which has heterodynecomponents; detecting said combined return beam to provide electricalsignals corresponding to said components; and processing said electricalsignals to produce an image of said tissue section.
 24. The methodaccording to claim 23 wherein said single beam of long coherence lengthlight represents a combination of each of said plurality of differentnon-overlapping wavelength bands have a coherence length greater thanone millimeter.
 25. The method according to claim 23 wherein said singlebeam defines a coherence function having peaks in accordance with saiddifferent wavelength bands, and said imaging beam transverses along afirst path from said first optics to said tissue and from said tissue tosaid second optics, and said reference beam transverses along a secondpath from said first optics through said optical modulator to saidsecond optics, in which the difference in length of said first path fromsaid second path is approximately equal to integer multiples of theseparation of the peaks of said coherence function.
 26. The methodaccording to claim 23 wherein said single beam from said light sourcedefines a coherence function having peaks in accordance with saiddifferent wavelength bands, said collected light of said imaging beamdefines a confocal response having a peak, and said interacting stepfurther comprising overlapping one of said peaks of said coherencefunction present in said reference beam with said peak of said confocalresponse.
 27. The method according to claim 23 wherein said scanning andfocusing step scans and focus said imaging beam along a focal plane insaid tissue, and said method further comprises the step of spatiallylimiting the light of said return beam to a region of said tissue insaid focal plane.
 28. The method according to claim 23 wherein saidprocessing step further comprises the step of demodulating saidelectrical signals to obtain data representative of said image of saidtissue section.
 29. The method according to claim 23 wherein said stepof producing a single beam of long coherence length light is carried outwith the aid of a plurality of light sources which produce saidplurality of different beams at each of said different wavelength bandsand optical elements for combining said plurality of different beamsinto said single beam.
 30. The method according to claim 29 wherein saidimage of said tissue section has an axial resolution dependent on atleast the number of said plurality of light sources.
 31. The methodaccording to claim 23 wherein said confocal optics provide a numericalaperture below 0.7.
 32. The method according to claim 23 wherein saiddifferent wavelength bands are in the infrared spectrum of light. 33.The method according to claim 23 wherein said imaging beam is scannedand focused to an illumination spot.
 34. The method according to claim23 wherein said scanning and focusing step is carried out with confocaloptics having at least an objective lens through which said imaging beamis focused below the surface of the tissue, said objective lens has anumerical aperture, and said tissue section imaged has an axialresolution in accordance with at least the numerical aperture of theobjective lens, and the number of different beams providing said singlebeam.
 35. The method according to claim 23 further comprising the stepof spatially limiting the combined return beam before said detectingstep is carried out.
 36. The method according to claim 23 wherein saidwavelength bands are equidistant from each other.
 37. The methodaccording to claim 23 wherein said long coherence length lightrepresents light with coherence length greater than one millimeter. 38.A system for imaging below the surface of an object comprising: aconfocal optical system for scanning said object and collecting lightfrom said a plurality of light sources operating at different wavelengthbands of long coherence length light which are combined into a singlebeam for illuminating said confocal optical system; means for splittingsaid single beam into an imaging beam for illuminating said confocaloptical system and a reference beam; means for modulating said referencebeam; means for combining said collected light from said object and saidmodulated reference beam; means for detecting the portion of saidcombined light representing a section to produce electrical signalsrepresentative of said section; and means for producing an image of saidsection of said object in accordance with electrical signals.
 39. Thesystem according to claim 38 wherein said long coherence length lightrepresents light with coherence length greater than one millimeter. 40.A method for producing an image of a section of an object using confocaloptics which scan and collect light from said object comprising thesteps of: combining into a single beam light from a plurality of beamsof different wavelength bands of long coherence length light; splittingsaid single beam into an imaging beam for illuminating said object viaoptical system and a reference beam; modulating said reference beam;combining light collected by said confocal optical system and saidmodulated reference beam; detecting the portion of said combined lightrepresenting said section of said object to produce electrical signalsrepresentative of said section; and producing an image of said sectionin accordance with said electrical signals.
 41. The method according toclaim 40 wherein said long coherence length light represents light withcoherence length greater than one millimeter.